Microbial protein production from methane via electrochemical biogas upgrading

Journal Pre-proofs Title: Microbial protein production from methane via electrochemical biogas upgrading Nayaret Acosta, Myrsini Sakarika, Frederiek-Maarten Kerckhof, Cindy Ka Y. Law, Jo De Vrieze, Korneel Rabaey PII: DOI: Reference:

S1385-8947(19)33040-2 https://doi.org/10.1016/j.cej.2019.123625 CEJ 123625

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

20 August 2019 24 October 2019 26 November 2019

Please cite this article as: N. Acosta, M. Sakarika, F-M. Kerckhof, C.K.Y. Law, J. De Vrieze, K. Rabaey, Title: Microbial protein production from methane via electrochemical biogas upgrading, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.123625

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Title: Microbial protein production from methane via electrochemical biogas upgrading.

Nayaret Acosta1+, Myrsini Sakarika1+, Frederiek-Maarten Kerckhof1, Cindy Ka Y. Law1, Jo De Vrieze1 and Korneel Rabaey1*

1Centre

for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links

653, B-9000 Gent, Belgium

Manuscript submitted to Chemical Engineering Journal

* Correspondence to: Korneel Rabaey, Ghent University; Faculty of Bioscience Engineering; Centre for Microbial Ecology and Technology (CMET); Coupure Links 653; B-9000 Gent, Belgium; phone: +32 (0)9 264 59 76; fax: +32 (0)9 264 62 48; E-mail: [email protected]; Webpage: www.cmet.Ugent.be. +Equally

contributed

1

Abstract Microbial protein (MP) can alleviate the increasing pressure of food demand on agriculture and our environment. For its sustainable production, feedstocks such as biomethane or (bio)hydrogen are needed. Here, we coupled biogas produced from agricultural waste directly with electrochemical biogas upgrading to subsequently produce MP from methane, hydrogen or a mixture thereof. Biogas was produced from co-digestion of pumpkin and pig manure at production rates of 0.73 ± 0.24 Lbiogas L-1reactor day-1 (59 % CH4) and 0.59 ± 0.29 Lbiogas L1

reactor

day-1 (50% CH4). The biogas was directed to the cathode of an electrochemical cell. At

current densities of 20 and 40 A m−2, CO2 removal efficiencies of 88 ± 14 % and 99 ± 1 % were achieved. Enrichments of MP (hydrogen- and methane oxidizing bacterial cultures) were cultivated on either raw biogas or gases obtained from the cathode (CH4, CO2, H2) and anode (CO2, O2) in batch mode with external supplementation of O2 and H2 when required. The best performance was obtained when the cathode off-gas was used in terms of biomass concentration (0.585 g CDW L-1), yield (0.150 g CDW g-1 COD), efficiency of COD conversion to protein (17%) volumetric biomass productivity (0.226 g CDW L-1 day-1) and volumetric protein productivity (0.181 g protein L-1 day-1). The protein content was similar when using anode and cathode off-gases (66.3 ± 7.3 % of CDW) with raw biogas resulting in a 6 % lower protein content. This proof of concept demonstrated that electrochemical biogas upgrading enables steering MP production. Keywords: agricultural residues; electrochemical biogas upgrading; single-cell protein; waste valorization.

2

1. Introduction Projections estimate an increase in the world population up to 9 billion people by 2050 [1]. This anthropogenic pressure on the world increases food requirements, which translates into great environmental impacts of agricultural expansion [2]. Agriculture contributes more than any other sector to certain forms of environmental degradation such as the conversion of natural habitats, deforestation, greenhouse emissions, etc. [3]. The use of synthetic nitrogen fertilizers has increased nearly 21-fold since 1950, but virtually all human-derived reactive nitrogen is lost to the atmosphere or receiving water bodies [4, 5]. Nutrient recovery from organic waste, especially agricultural waste, is a method for preventing environmental decay while enabling increased food supplies, by being the only method for large-scale protein production that does not require a concomitant increase in energy usage [6]. Besides, it may be the most effective method for producing animal and human food from lignocellulosic materials that are of little nutritional value and are, therefore, used as fuel [7]. A promising method to valorise agricultural waste for feed production is through microbial protein (MP). The term “MP” is used to describe protein contained in the cells of various microorganisms such as yeast, fungi, algae, and bacteria, which can be disrupted (e.g. Marmite, Unilever, UK) or consumed as such (Feedkind®, Calysta, USA). MP is conventionally produced on edible sugars (QuornTM, Marlow Foods, UK) or natural gas (Feedkind®; Uniprotein®, Unibio A/S, Denmark), while their production in inexpensive carbon sources, such as potato processing water (ValProMic, Avecom, Belgium) has recently been initiated. MP production has the potential to replace 10-19 % of the crop-based protein-rich feed, leading to significant environmental benefits, such as decreased agriculture-associated

3

greenhouse gas emissions (7 %), nitrogen losses (8 %) and cropland use (6 %) [6]. This brought about the commercialization of MP composed of methane oxidizing bacteria (MOB; Feedkind®, Calysta, USA), as well as pilot-scale units producing MP by hydrogen oxidizing bacteria (HOB; Power-to-Protein concept, Belgium and Netherlands). MP is produced by using pure cultures (e.g. QuornTM), synthetic communities (e.g. Feedkind®) or mixed cultures (e.g. ValProMic). A variety of agricultural and industrial wastes, as well as petroleum derivatives (e.g. natural gas and alkanes), have been investigated as feedstock for MP production [7]. An interesting agricultural waste for re-valorization is pumpkin. Yearly, more than 5.9 million tonnes of pumpkins are thrown into landfills in the United States, while in the United Kingdom alone, 3.6 million ton pumpkin end up in the garbage [8]. Even though pumpkin waste is considered a seasonal issue, the sheer volume of production has prompted the U.S. Department of Energy to include pumpkin in the projects related to integrated biorefineries for which AD is the main technology [9].To improve methane and biogas yields, co-digestion with animal manure is the most commonly strategy used [10]. Manure provides nutrients, buffer capacity and microorganisms that help to stabilize the AD process [10-12]. Nevertheless, there are risks associated with the direct use of (agro-)industrial waste for the production of MP [13]. The main risk is the potential bioaccumulation of toxic compounds, such as pesticides, heavy metals and chlorinated hydrocarbons, present in these waste streams. The bio-accumulation of such pollutants in the MP might impede consumption of this product. Therefore, it is necessary to convert the organic wastes into a non-toxic material that is easy to handle and still fully accessible to the microorganisms. An attractive approach is the conversion of organic waste into a gaseous stream such as biogas. In that respect, anaerobic digestion is an established technology to treat organic biomass (e.g., agricultural 4

residues), with conversion into a gas stream with high energy value, i.e., biogas as a mixture of predominantly CH4 and CO2 [14]. This opens the possibility to evaluate two different carbon sources for MP production together or separately. Commercial biogas upgrading technologies are already available. They are based either on physicochemical processes (absorption and adsorption) or biological processes, which involve the supplementation of reduced substrates by electrochemical means (e.g., H2) to biologically convert CO2 to CH4 in situ [15]; or ex situ [16] with the aid of methanogens [17, 18]. Electrochemical biogas upgrading can efficiently separate CO2 from CH4 [16] using renewable energy (e.g., solar) and has the advantage of producing H2 and O2 [19-21], needed for MP production, with the former being essential in case that CO2 is the carbon source [22]. Also, hythane, a mixture of methane and hydrogen, produced in electrochemical systems, might be beneficial for MP production [20]. The key objective of this research was to investigate whether raw biogas, upgraded biogas or separated CO2 form the best substrate for MP production via an electrochemical upgrading process. To our knowledge, this is the first time that electrochemical biogas upgrading is coupled directly with the production of MP. Furthermore, the combination of HOB and MOB has been evaluated in a single reactor, with the aim to smultaneously valorise all the produced gases. We tested a real feedstock for AD, in this case, pumpkin and pig manure. First, the separation of CO2 and CH4 from the real biogas was achieved in an electrochemical cell. Next, these gases were used as the main feedstock for high-quality HOB and MOBbased MP production. The combination of both protein products reflects the integrated valorisation of waste streams towards renewable feed/food products with renewable electricity as the sole input.

5

2. Materials and methods 2.1

Anaerobic co-digestion of pumpkin and pig manure

2.1.1 Pumpkin and cattle manure characterization Pumpkin was obtained from a local market (Ghent, Belgium). Pig manure was obtained from a local farm in West-Flanders (Belgium). Both feedstocks were characterized in three technical replicates (Table 1). Pumpkin was stored at -20 ˚C while manure was stored at 4˚C. Prior feeding, the pumpkin was unfrozen and mixed with the manure (24- 48 h before feeding). Table 1 Characterization of feedstock used in anaerobic digestion. Mean values ± standard deviation (n=3) are shown. TS

VS

COD

TKN

TAN

%

%

gL-1

gL-1

gL-1

8.91

8.02

101

1.82

1.18

± 1.40

± 1.42

± 4.36

± 5.50

1.65

1.19

21.17

± 0.01

± 0.09

± 0.76

pH

N

C

H

O

wt.%

wt.%

wt.%

wt.%

5.9

2.13

43.39

6.02

43.89

± 0.03

± 0.02

± 0.03

± 0.32

± 0.02

± 0.36

1.83

1.33

7.77

1.2

41.93

5.24

34.35

± 6.56

± 0.04

± 0.01

± 0.04

± 0.44

± 0.11

± 0.37

Feedstock

Pumpkin

Pig manure

TS: total solids; VS: volatile solids; COD: chemical oxygen demand; TKN: total Kjeldahl nitrogen; TAN: total ammonium nitrogen. Oxygen content was obtained by difference.

2.1.2 Anaerobic digester setup and procedures Co-digestion of pumpkin with pig manure was carried out in two glass Schott bottles (4 L working volume, 5L total volume) at mesophilic conditions (35 ± 1˚C) in a wet mass ratio 10/1 pumpkin/manure to assure a C/N ratio of 20/1. They were placed inside an incubator with automatic rotating shaking (20 rpm) (New Brunswick Scientific, USA) (Figure 1.a). The organic loading rate (OLR) was increased stepwise from 1 up to 2 g VS L-1 d-1 during the 6

first two weeks by increasing the amount of feedstock fed in the reactors. The feeding was performed three times per week. Biogas production and composition were monitored three times per week before connecting into the electrochemical system and then on a daily basis. The pH was monitored with every feeding and volatile fatty acids (VFA), TS and VS from the effluent were analysed on a weekly basis.

2.2

Electrochemical biogas upgrading

2.2.1 Experimental setup The setup was composed of a two-chambered electrochemical cell (EC) with two Perspex frames, joined by an anion exchange membrane (Type II, Fujifilm Manufacturing, The Netherlands) based on Veerbeck et.al, [16] (Figure 1.b). Both electrode chambers had a working volume of 0.2 L (internal dimensions: 5 × 20 × 2 cm3). The cathode was a stainlesssteel wire mesh of 5 × 20 cm2 exposed working area and 564 μm mesh width (Solana, Belgium), equivalent to a projected surface area of 100 cm2. The anode was iridium mixed metal oxide (Ir MMO) coated titanium-electrode mesh (TiO2/IrO2, 0.35/0.65, Magneto Special Anodes, The Netherlands). Both electrodes were 0.5 cm away from the anion exchange membrane (AEM) to minimize ohmic resistance, a turbulence promoter mesh (ElectroCell, Denmark) was used to prevent contact between the surface of the electrodes [16]. A solution of 0.25 M HEPES buffer worked as catholyte (pH 8.12), while 0.25 M sodium sulphate (pH 1.5) was used as anolyte.

2.2.2 Electrochemical cell operation The anolyte and catholyte were recirculated (0.78 L h-1) from a 0.5 L Schott bottle (0.4 L working volume) to their respective compartment with the help of a peristaltic pump (Watson Marlow, United Kingdom). The length of tubing (Tygon, USA) that connected all the 7

cathode compartment was double than the tubing length from anode compartment (360 cm cathode – 180 cm anode;  4 mm) to improve gas/liquid contact of biogas and HEPES buffer. The temperature was maintained at 20 ± 1 °C in a temperature-controlled room. The biogas produced by the AD reactors was connected to the cathode through a gas counter (Ritter, Germany), with a maximum capacity of 500 mL h-1 and a glass gas trap. Two current densities were tested to assess EC performance for CO2 removal, i.e., 20 and 40 A m-2, to assure a balance between maximum removal and high current efficiency. Calibrated semiopen gas columns were used to measure and store the gases produced from the cathode and anode compartments (Figure 1.b). Gas samples were taken at least every two days; the samples points were from the entrance of the biogas into the cathode, the outside of cathode compartment, the outside of the anode compartment and from the outside of the recirculation bottles (Figure 1.b). The pH was measured in the liquid samples from the recirculation bottles from the cathode and anode compartments.

2.3

Microbial protein production from biogas, cathode off-gas and

anode off-gas 2.3.1 Enrichment of hydrogen and methane oxidizing bacteria Methanotrophic (MOB) and hydrogenotrophic (HOB) enrichments were cultivated under non-axenic conditions in 1.2 L Schott bottles, sealed with butyl rubber stoppers, with a liquid volume of 0.2 L and a gas phase volume of 1 L. The MOB enrichment was performed using ammonium mineral salts (AMS) [23] and air supplemented with c.a. 20 % CH4 (99.995 % purity). The HOB enrichment was performed using a medium for chemo-lithotrophic growth (DSMZ medium 81) [24] under a headspace gas composition of c.a. 40 % H2, 40 % CO2 and 20 % O2. The initial inoculum for the MOB enrichment comprised c.a. 10 g (in wet weight, 8

WW) compost originated from Ghent University (campus Coupure, Ghent, Belgium) for the MOB enrichment. The HOB enrichment was initiated using c.a. 10 g WW of sediment collected from a former oyster pond of a river estuary (Yzer, Nieuwpoort, Belgium). The latter was selected, due to the abundance of hydrogen oxidizers in such environments [25]. The bottles were incubated on a rotary shaker (120 rpm) at 28°C and were regularly recultivated (1-2 times per week for 2 months; 10 transfers in total with an average interval of 7 days) through transferring 10 % (v/v) in fresh media.

Figure 1 Overview of the integrated process scheme involving anaerobic digestion (a), electrochemical biogas upgrading (b) and microbial protein production from biogas, cathode and anode off-gas (c). Light-blue dotted lines correspond to biogas, red dotted lines to cathode off-gas and dark-blue dotted lines to cathode off-gas.

2.3.2 Microbial protein production using hydrogen- and methane oxidizing enrichment cultures Batch experiments were performed in opaque serum vials with a total volume of 120 mL, sealed with butyl rubber stoppers. The cultivation medium was AMS supplemented with the 9

vitamin solution of DSMZ medium 81 (Table A, supplementary information). The working volume was 20 mL (10 % inoculation, i.e., 18 mL medium and 2 mL of inoculum or distilled water for the control treatment), whereas the remaining 100 mL was headspace. All inocula were composed of a 1:1 VSS ratio of HOB and MOB enrichment cultures. Fresh gas was collected from the anaerobic digester, cathode off-gas as well as anode off-gas (Table B, supplementary information) before the conduction of the MP production experiments. All the gasses used in the experiments were supplied through manual flushing of each serum vial. Next, 20 mL O2 was injected using a sterile 50 mL syringe. Oxygen was resupplied according to the need to maintain non-toxic O2 levels (>40 % v v-1) [26], as well as non-limiting concentrations (<0.01 % v v-1) [27]. The procedure differed for the serum vials supplied with the anode off-gas: the vials were initially flushed with N2 for 10 minutes, next 40 mL of the gas was replaced with anode off-gas manually (to avoid O2 toxicity effects [26]), and finally 40 mL of hydrogen gas was added, as an energy source for HOB growth. The serum vials were subjected to overpressure at the start of each experiment (up to c.a. 50 kPa). All gases were filter-sterilized (0.20 µm PTFE filters, Sartorius) to avoid contamination. The gas composition was analyzed daily; the CDW was quantified at the beginning and the end of the experiment; and the protein content was determined at the end of each experiment. The parameters analyzed were biomass concentration (g CDW L-1), biomass yield (g CDW g1

COD), volumetric biomass productivity (g CDW L-1 day-1), protein content (% CDW) and

volumetric protein productivity (g protein L-1 day-1), considering the initial and final point of each test. Experiments were performed at 28°C on an orbital shaker with a mixing intensity of 120 rpm, under aseptic conditions in quadruplicates and mean values ± standard deviation are presented. 10

2.3.3 Conversion efficiency of biogas, cathode off-gas and anode off-gas to microbial protein The conversion efficiency of chemical oxygen demand (COD) to MP was calculated based on Equation 1:

𝐸𝑓𝑓𝑖𝑐𝑒𝑛𝑐𝑦 (%) =

𝐶𝑂𝐷 𝑚𝑖𝑐𝑟𝑜𝑏𝑖𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 [𝑔𝐶𝑂𝐷 𝐿 ―1] 𝐶𝑂𝐷 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 [𝑔𝐶𝑂𝐷 𝐿 ―1]

× 100

Equation 1

where ―1] 𝐶𝑂𝐷𝑚𝑖𝑐𝑟𝑜𝑏𝑖𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 [𝑔𝐶𝑂𝐷 𝐿 ―1] = 𝐶𝑂𝐷𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑𝑠 [𝑔𝐶𝑂𝐷 𝑔𝑀𝑃 ∙ 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 [𝑔𝑀𝑃 𝐿 ―1]

𝐶𝑂𝐷𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑𝑠 [𝑔𝐶𝑂𝐷 𝐿 ―1] =

𝐴𝐴1 + 𝐴𝐴2 + … + 𝐴𝐴𝑛 𝑛

―1] [𝑔𝐶𝑂𝐷 𝑔𝑀𝑃

Equation 2

Equation 3

The COD of the amino acids (AA) was calculated based on the assumptions that (1) the amino acids contained are essential for human nutrition (i.e., histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) [28], and that (2) the amino acids are present in equal amounts (weight basis). The average COD of the MP was then calculated as 1.55 g COD g-1 MP based on the theoretical COD of the individual amino acids. Finally, the consumed COD was calculated as follows: 𝐶𝑂𝐷𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 [𝑔𝐶𝑂𝐷 𝐿 ―1] = 𝐶𝑂𝐷𝑔𝑎𝑠 𝑝ℎ𝑎𝑠𝑒,

2.4

𝑖𝑛𝑖𝑡𝑖𝑎𝑙

[𝑔𝐶𝑂𝐷 𝐿 ―1]– 𝐶𝑂𝐷𝑔𝑎𝑠 𝑝ℎ𝑎𝑠𝑒, 𝑓𝑖𝑛𝑎𝑙 [𝑔𝐶𝑂𝐷 𝐿 ―1]

Equation 4

Analytical techniques

Total solids (TS), volatile solids (VS), total suspended solids (TSS), VSS, total Kjeldahl nitrogen (TKN) and COD were analyzed using Standard Methods [29], while the VFA concentration (C2 to C8) was measured via gas chromatography (GC-2014, Shimadzu®, The 11

Netherlands) based on Andersen et. al., [30]. The pH was measured using a pH meter C532 (Consort, Turnhout, Belgium). Crude protein was analyzed using the Markwell method [31], using bovine serum albumin as a standard (analytical triplicates). The samples for protein analysis (1 mL suspension) were centrifuged at 20,817 rcf (14,000 rpm), washed once with phosphate buffer and stored at -4°C. The gas composition was measured by means of a compact-GC gas chromatograph (Global Analyser Solutions, Breda, The Netherlands). In the first channel, CH4, O2, N2 and H2 (Porabond Q pre-column and Molsieve 5A column) were detected, while in the second channel (Rt-QS-bond column and pre-column) CO2 was measured. The detection limit was 100 ppmv for each gas, using a thermal conductivity detector.

2.5

Calculation and analysis of data

The R language for statistical programming (version 3.6.1), was used for statistical analysis (http://www.r-project.org) of the molar percentage of biogas cathode- and anode-off gases. First, normality was checked by a Shapiro-Wilk Normality test and visually using Q-Q plots. Homoscedasticity was analyzed with a robust Levene-type/Brown-Forsythe test (R package lawstat, v 3.3). If normality and homoscedasticity were not rejected, a Student t-test was used to evaluate significant differences. If normality and homoscedasticity were rejected, the Wilcoxon rank sum test was used instead.

3. Results 3.1

Anaerobic co-digestion of pumpkin and pig manure

Two digesters were operated in parallel using a 10/1 pumpkin/manure blend. Reactors 1 and 2 (i.e., R1 and R2) showed a similar biogas and methane production rate during the first 50 12

days of operation (Figure 2), during the last days of the experiment more variability was observed. The biogas production rates were 0.79 ± 0.37 L L-1reactor day-1 for R1 (54 % CH4) and 0.49 ± 0.31 L L-1reactor day-1 in R2 (48 % CH4). When the biogas from R1 was connected to the EC, the production rate was 0.84 ± 0.42 L L-1reactor day-1. The R1 produced up to 29 % more CH4 and CO2 than R2. Sulphide was not detected during the experiment (detection limit 500 ppmv). While the CO2 yields from R2 remained fairly constant, 126 ± 50 L kg-1 VS during the II period (OLR 2 g VS L-1 day-1), the CO2 from R1, especially while it was connected to the EC cell, varied greatly with an average of period II and III of 144 ± 59 L kg-1 VS, compared to the period III (connected to EC), 148 ± 83 L kg-1 VS (Figures A and B, supplementary information). Methane yield was recovered in R1, after the partial failure of both reactors (after day 50). This was not the case in R2, where the methane yields remained low for the rest of the experimental period.

13

Figure 2 Methane and carbon dioxide production rate in the two anaerobic digesters using pumpkin and manure as feedstock in mesophilic conditions. Period I correspond to start-up with organic loading rate (OLR) of 1 g VS L-1 day-1, period II and III have an OLR of 2 g VS L-1 day. In period III, the biogas from R1 was directly connected to an electrochemical cell for biogas upgrading.

The TKN concentrations remained similar between R1 and R2 during the experiment, below assumed toxic levels (Figure C, supplementary information). The pH and VFA concentration remained similar during the first 50 days and then varied afterwards with stable pH values in R1, (pH = 6.95 ± 0.69; VFA = 68 ± 61 mg COD L-1), while R2 had a lower pH (6.67 ± 0.54) and slightly higher VFA (100 ± 90 mg COD L-1. The pH was controlled with addition of NaOH (1M) to a pH 7.2 after each feeding, when necessary. While R1 recovered the pH in the long term, R2 maintained a low pH until the end of the experiment (Figure C, supplementary information). When the biogas from R1 was connected to the EC, the average pH was 7.30 ± 0.34 and the VFA concentration was 106 ± 46 mg COD L-1. The average VS removal efficiency was 84 ± 4 % and 85 ± 4 % for R1 and R2, respectively. The COD removal efficiency was on average 69 ± 7 % in R1 and 67 ± 10 % in R2 (Figure C, supplementary information).

3.2

Electrochemical biogas upgrading

The biogas produced from digester R1 was connected directly to the electrochemical cell to separate CO2 from the biogas stream. The composition of the biogas going into the cathode compartment is shown in Figure 3. Principally, the EC and the current densities applied were designed for the total available biogas flow from both reactors, but a proper connection was not possible which relates to practicalities of creating small reactors at laboratory scale evenly matched in terms of production. The biogas from R1 was connected to the EC because it produced higher yields than R2. As such, the EC was as such over-dimensioned however 14

process optimization was not the scope here. The composition of CH4 and CO2 varied periodically, due to the fed batch system, three times per week with more CO2 on the feeding day and late increase of CH4 as days passed. However, the mol percentage of CO2 and CH4 were not significantly different (p>0.05) at 20 A m-2 compared to 40 A m-2. Meanwhile, in the cathode off-gas there were significant differences of molar percentage of CH4 (p<0.003), H2 (p<0.0002) and CO2 (p<0.01) between 20 and 40 A m-2 (Figure 3). The molar percentage of O2 in anode-off gas was significantly different at the two current densities applied (p<0.03) however the CO2 molar percentage did not have significant differences (p>0.055) (Figure 3). At 20 A m−2, an average CO2 removal efficiency of 88 ± 14 % was achieved, calculated based on the volume of CO2 extracted, relative to the volume of CO2 injected (Figure 4). At this current, between day 8 and 10, the pH from the catholyte was controlled with 1M H2SO4 to achieve a pH of 8.1, to assure that the CO2 from the biogas was converted mainly into the HCO3- form instead of CO32-. In the short period where the pH was controlled, a removal efficiency of 65 ± 5 % was obtained. The current efficiency (CE), calculated as mol bicarbonate-carbonate extracted per mol electrons supplied, was 39 ± 27 % at 20 A m−2. When not taking into account the period where the pH was controlled, the CE was 3 % higher, 42 ± 28 %. The pH was not controlled further on. Average pH values of 10.02 ± 2.20 and 11.48 ± 2.12 were obtained at 20 and 40 A m-2, respectively, in the catholyte, while the pH in the anolyte was 1.90 ± 0.73 at 20 A m-2 and 2.21 ± 0.16 at 40 A m-2. There were two peaks in the CO2 flux, i.e., on day 71 and 78. This coincided with the manual feeding of the reactors, which might have led to rapid degradation of the biomass leading to high CO2 production rates. During these two days, the CO2 input was high enough to achieve a theoretical current efficiency of 100 % (Figure 3). However, the CE on these points was 91 and 100 %, with CO2 removal efficiencies of 81 and 90 %, respectively. At 40 A m−2, the 15

removal efficiency reached 99 ± 1 % on average, with an average CE of only 10 ± 5 %, due to the low CO2 flux from the biogas input relative to system size. An earlier study [16] already demonstrated on a synthetic gas that CE values can be very high, and this was not the objective of the present study.

Figure 3 Biogas injected to the cathode in the electrochemical cell (a), cathode off-gas (b) and anode off-gas (c) resulting from the biogas upgrading by electrochemical means, presented in mol %.

The extraction of CO2 from the biogas stream occurred through dissolution into carbonate and bicarbonate ions, and subsequent transfer to the anode compartment across the AEM. At 20 A m−2, a water flux of 3.0 L m−2 day−1 from the cathode compartment was observed, while at 40 A m−2, the water flux increased to 3.7 L m−2 day−1, indicating a relationship between the water transport and the current applied. The excess of liquid from the anode was discarded,

16

while a fresh solution of HEPES buffer was added in the cathode side, thus, balancing the volume in the compartments without impacting the system in a major way.

Figure 4 Carbon dioxide flux from the biogas stream in L CO2 extracted m-2day-1 and mol CO2 day-1, with the corresponding removal efficiency (RE) and current efficiency (CE). Two current densities where tested, i.e., 20 A m-2 (white background) and 40 A m-2 (light grey background).

The CO2 was successfully removed from the biogas during the experimental period, with exception of the start-up phase and during the period in which the pH was controlled in the cathode compartment (day 8 to 11). The cathode off-gas was mainly composed of H2 (50 ± 10 %) and CH4 (37 ± 9 %) when the current density was 20 A m-2. The H2 content increased up to 71 ± 7 %, while the CH4 content decreased to 21% when the current was 40 A m-2 showing the ability of electrochemical upgrading to fine-tune gas composition. In the anode off-gas, the composition was mainly CO2 and O2 in a % mol ratio close to 1:1, being 50 ± 15% CO2 and 47 ± 15 % O2 at the lower current density (Figure 3). The O2 content increased due to water electrolysis. In terms of mass balance, an average of 49 ± 22 % of the CO2 from

17

the biogas ended up in the anode off-gas at 20 A m-2, 12 ± 14 % in the cathode-off gas and 42 ± 23 % is assumed to be dissolved in the catholyte or loss. On average, 75 ± 23 % of the CH4 was recovered in the cathode off-gas at the same current density. When the current density was increased up to 40 A m-2, 98 ± 7% of the CO2 in the biogas ended up in the anode off-gas and 1 ± 6 % on average went to the cathode off-gas column. A similar high recovery of CH4 in the cathode off-gas was obtained, reaching 100 ± 2 % on average.

3.3

Microbial protein production

The production of microbial protein using either biogas or the electrochemically upgraded gas streams was demonstrated by cultivating the HOB and MOB in batch mode. Overall, the best performance in terms of biomass concentrations, yields, COD conversion to protein, as well as volumetric protein productivity, was obtained when the cathode off-gas was supplied. After 2.58 ± 0.01 days of growth, the biomass concentration when cathode off-gas was provided reached values 39 % and 152 % higher compared to the use of biogas or anode offgas, resulting in biomass yields of 51 % and 47 % higher respectively (Table 2). The COD (i.e., CH4 and H2) conversion efficiency to protein was 9%, 17% and 14% for biogas, cathode off-gas and anode off-gas, respectively. Table 2 Batch growth performance parameters of the combination of enriched methane and hydrogen oxidizing bacteria used for microbial protein production in serum vials supplied with biogas, cathode off-gas and anode off-gas. Hydrogen gas was supplemented along with the anode off-gas as energy source to enable the growth of hydrogen oxidizing bacteria. Oxygen was supplemented to cathode off-gas and biogas. Mean values ± standard deviation (n=4) are presented. Gas feedstock

Biomass concentration (g CDW

L-1)

Yield (g CDW g-1 COD)

Volumetric biomass productivity (g CDW

Biogas

0.420 ± 0.065

0.100 ± 0.018

L-1

Protein content (%CDW)

Volumetric protein productivity (g protein L-1day-1) *

day-1)

0.162 ± 0.025

60.47 ± 8.39

0.097 ± 0.017

18

Cathode off-gas

0.585 ± 0.064

0.150 ± 0.019

0.226 ± 0.025

66.60 ± 7.61

0.181 ± 0.036

Anode offgas

0.232 ± 0.046

0.102 ± 0.024

0.091 ± 0.018

66.06 ± 9.18

0.080 ± 0.017

* assuming the protein content is constant throughout the growth L= volume of liquid

The total COD consumed was 4.24, 3.91 and 2.27 g COD L-1 for biogas, cathode and anode, 88.2, 70.1 and 0% of which originated from CH4 (Table 3). The substrate oxidation rate was 1.64, 1.51 and 0.89 g COD L-1 day-1. The protein content was comparable for the cases where cathode off-gas and anode off-gas were supplied (66.1-66.6 %CDW), with biogas presenting a 6% lower value, while the volumetric protein productivity was up to 107% higher when cathode off-gas was supplied. Table 3 Substrate consumption (g COD L-1) and substrate oxidation rate (g COD L-1 day-1) during the batch tests. Mean values ± standard deviation (n=4) are shown. Units Methane (CH4) Hydrogen (H2)

g COD L-1

Total (CH4 + H2) Methane (CH4) Hydrogen (H2) Total (CH4 + H2)

g COD L-1 day-1

Biogas

Cathode off-gas

3.74 ± 0.13 0.49 ± 0.04 4.24 ± 0.12 1.44 ± 0.05 0.19 ± 0.01 1.64 ± 0.05

2.74 ± 0.16 1.18 ± 0.24 3.91 ± 0.19 1.06 ± 0.06 0.46 ± 0.09 1.51 ± 0.07

Anode off-gas N.A. 2.27 ± 0.17* 2.27 ± 0.17* N.A. 0.88 ± 0.06 0.88 ± 0.06

N.A. = not applicable L= liquid volume *Hydrogen was supplemented along with the anode off-gas as energy source to enable the growth of hydrogen oxidizing bacteria.

Regarding the gas consumption rate (Figure D, supplementary information), CH4 and H2 are linearly consumed by MOB and HOB, respectively, in the cases were biogas or cathode off19

gas is supplemented. In these experiments, the CO2 increased in the gas phase, as a result of the respiration of the methanotrophs. Conversely, both CO2 and the supplemented H2 were almost completely eliminated when the anode off-gas was supplied, with end values of 0.051 ± 0.014 mmol CO2 and 0.023 ± 0.022 mmol H2.

4. Discussion 4.1

Electrochemical biogas upgrading enables effective separation

of CH4 and CO2 Biogas production from AD of pumpkin with manure was produced for 117 days. At the end of the experiment, from day 67 onwards, a higher variation in yields in R1 and R2 was observed, and this could be attributed to the use of unfrozen feedstock that was defrosted at least one week in advance from the feeding which led to self-degradation of the feedstock. This variation was not noticed immediately, because the effect of the feedstock was not monitored continuously. The pH was controlled with addition of NaOH (1M) to a pH 7.2 after each feeding; while R1 recover the pH in the long term, R2 maintained a low pH until the end of the experiment (Figure C, supplementary information). The present study demonstrated that a real biogas stream (with variable production and composition) can be separated successfully, which showed the robustness of an electrochemical system to keep CO2 removal close to 100%, regardless the composition of the inlet biogas. The current efficiency was low, due to the insufficient load of CO2 injected to the EC. The EC was designed for the available biogas flow from the two AD reactors, but it was not possible to connect both digesters continuously to the EC through the gas counter.

20

Kokkoli et al. [32] performed a similar experiment for a shorter period (5 days), lower current density (0.32 – 0.39 A m-2) and lower volume of biogas injected (0.36 L in total), reaching a removal efficiency of 99 % of CO2 from a synthetic biogas stream. Another study found a CO2 removal efficiency close to 100 % during the first 4 days of operation when the injected concentration of CO2 was kept below 10 % from the total gas composition until the end of the experiment. The cell potential in this case was 1.2 V with a current density of 1.3-1.7 A m-2 [33]. Higher current densities (0 - 400 A m-2) and biogas loads proved that CO2 removal from the biogas stream was mainly due to the current applied to the cell [16]. With a pH as high as 11.5 in the catholyte, 99% of the CO2 injected from biogas will become CO32–, but at lower pH, HCO3– becomes dominant up to 99.9% when pH is 8.1. The speciation of carbonate components is difficult to measure, due to the interactions with other ions, and because of the nature of the catholyte, which is composed of HEPES buffer. This large buffer was used to avoid the electro-migration of the buffer towards the anode [33], avoiding pH fluctuations despite the injection of CO2 [34]. As CO32- is a bivalent anion, the theoretical maximal CO2 flux through CO32– is only half of the maximum flux via HCO3– [16]. Veerbeck et.al., [16] calculated a CO2 mass closure of 88 ± 3% in a three hour experiment with similar electrochemical condition as this study. Due to the fact that this study operated longer and with variable biogas production and composition it could be assumed that part of the CO2 was embedded in the catholyte in the form of carbonate. Also, small loses could have be product of CO2 loss in the anode chamber and measurement errors. Part of the CE decrease at higher current density is thus most likely linked to the higher relative contribution of CO32– in the charge balance [16], and also the low quantity of CO2 injected into the system explains the low CE in this experiment.

21

4.2

Microbial protein from raw or electrochemically upgraded

biogas The biomass concentrations and biomass volumetric production rate were comparable to other studies conducting batch tests for MP production, but remained at low levels, in comparison to studies working with continuous or semi-continuous systems (Table 4). The biomass concentrations achieved in the present study are up to 2.2 times the concentration achieved by Rasouli et al. [35], who cultivated M. capsulatus in batch mode. The concentrations are considerably lower than for continuous mode production systems though. Mattasa et al. [22] reported biomass levels up to 11.2 g CDW L-1 in a sequential batch reactor (SBR) for HOB production and Sheehan and Johnson [36] up to 12.8 g CDW L-1 in a continuous reactor for MOB production (at solids retention time of 10 h and 5.35 h, respectively). Nevertheless, this is a consequence of the amount of gas used relative to amount of medium in these batch tests. The volumetric biomass productivities remained at low levels as well, however, a comparison with studies using other modes of cultivation is not valid since, apart from the different performance of the microbiota according to the cultivation mode, in the present study the lag phase is also taken into account, which is not the case in continuous operation. Table 4 Comparison of results obtained in this study with other studies regarding microbial protein production based on methanotrophic and/or hydrogen oxidizing bacteria. Mean values ± standard deviation is presented.

Strain/ microbial culture

Mixed Methylococcus capsulatus

Gas phase composition

35% CH4; 23% air; 43% O2 40% CH4; 60% air

Cultivation mode

Biomass concentration g CDW L-1

(number of observations)

continuous (n=16) batch (n=3)

Yield g CDW g-1 COD

Volumetric Volumetric biomass protein productivity productivity g CDW day-1

L-1

g protein day-1

L-1

Crude protein content

Reference

% CDW a

1.61-12.8

0.154

17.4-57.4

10.1-33.1

57.7 b

[36]

N.A.

N.A.

N.A.

N.A.

40.0-51.5

[37]

22

60% CH4; 30% O2; 10% CO2

Methylococcus capsulatus Mixed (dominated by Sulfuricurvum sp.)

65% H2; 20% O2; 15% CO2

Mixed Mixed (dominated by Sulfuricurvum sp.)

45% H2; 15% O2; 40% CO2

Mixed (dominated by Methylococcales and Methylophilales)

Mixed

Mixed

Mixed

aassuming

batch (n=3) continuous (n=35)

0.264

N.A.

1.15

0.602

52.5

3.75 ± 0.04

0.280 ± 0.010

9.00 ± 0.36

6.39 ± 0.26

71.0 ± 5.0

[35]

[22] sequential batch (n=N.A.)

11.2 ± 0.2

0.073 ± 0.007

1.87 ± 0.29

1.24 ± 0.19

66.0 ± 5.0

fed-batch (n≥6)

3.74-4.44

0.1300.170

1.72-1.85

N.A.

N.A.

[38]

N.A.

N.A.

28.8% e

[39]

33% CH4; 66% O2 c

0.190 ± 0.020

33% CH4; 66% O2 d 20% CH4; 66% O2; 13% CO2; H2S traces (biogas) d 31% CH4; 66% O2; (biologically upgraded biogas) d 32% CH4; 18% O2; 11% CO2; 18% H2 (biogas) 26% CH4; 15% O2; 35% H2 (cathode offgas) 15% O2; 5% CO2; 23% H2 (anode offgas)

0.178 ± 0.013

batch (n=3)

N.A.

0.218 ± 0.020

0.206 ± 0.005

batch (n=4)

0.420 ± 0.065

0.100 ± 0.018

0.162 ± 0.025

0.097 ± 0.017

60.5 ± 8.4

0.585 ± 0.064

0.150 ± 0.019

0.226 ± 0.025

0.165 ± 0.036

66.6 ± 7.6

0.232 ± 0.046

0.102 ± 0.024

0.091 ± 0.018

0.080 ± 0.017

66.1 ± 9.2

This study

that the protein content remains constant through time

bn=1 cusing

centrifuged-filtered digestate as nitrogen source extracted ammonium as nitrogen source ebased on nitrogen content of biomass (N%·6.25); values for individual experiments were not given N.A. = not available delectrochemically

The best results were obtained when providing the cathode off-gas, i.e., the mixture of CH4, and H2, while CO2 is generated in situ by the metabolism of MOB (catabolic oxidation of CH4 23

to CO2). This could be attributed to the fact that the composition of the cathode off-gas – containing the energy and carbon source for both MOB (CH4) and HOB (H2/CO2) – enabled the active growth of both MOB and HOB, therefore increasing the overall efficiency of the system. Furthermore the higher initial H2 content of cathode off-gas (35%) compared to the supplemented H2 in the biogas (18%) might have contributed to a ratio of H2:O2:CO2 that is more conducive to HOB growth (Figure D, supplementary information), compared to the case where biogas was provided [40]. The protein content achieved in this study (60.5-66.6% CDW) is in line with the available literature (Table 4). Specifically, it favourably competes with the results obtained for MOB, reaching values up to 2.3 times higher (66.6 % CDW compared to 28.8% CDW), while it is comparable to sequencing batch reactors producing MP based on HOB (Table 4). The comparison with conventional protein supplements is favourable, with fishmeal having similar protein content (i.e., 68 % DW) [41] and soybean meal containing c.a. 1/3 less crude protein (i.e., 48 % DW) [42], compared to the MP produced in this study. The average protein content of a commercial bacterial meal is 70.3% CDW [41], which is only slightly higher (6%) than in the present study. Bacterial MP has some additional properties (compared to soybean and fishmeal) that are beneficial for the organism consuming it [43]. Several studies documented the beneficial effects of MP on feed conversion when used in aquaculture [44], meat quality when used as feed for chicken [45] and health of monogastric animals including broiler chicken, pigs, mink and fox [41]. Mixed cultures can create added value for MP production compared to pure cultures. As an example, the added value of the presence of heterotrophs in methanotroph-dominated cultures has been highlighted [46, 47]. Similarly, the synergism between methanotrophs and algae has been demonstrated [48]. Specifically, heterotrophs have been reported to stimulate the methane oxidation rate of Methylomonas methanica during batch tests, with the effect

24

being more significant at an increased number of heterotroph cells [49]. Furthermore, the continuous production of microbial protein from natural gas using Methylococcus capsulatus requires a co-culture with heterotrophs belonging to the genera Ralstonia, Brevibacillus and Aneurinibacillus to ensure process stability [46]. These genera consistently invaded pure cultures of M. capsulatus during continuous industrial production of microbial protein [46], and are now inoculated in the methanotrophic monoculture [50]. Pure cultures could lead to the accumulation of methanol [51], organic acids as well as free amino acids (and other cell lysates) [52], as products of the methanotrophic metabolism, which has been reported to be a cause of growth inhibition of MOB [53]. The flanking community thus scavenges these metabolites, effectively stabilizing the production process. In the present study, the presence of HOB and MOB in the same inoculum resulted in an increased efficiency compared to the case of inoculating only with MOB or HOB. Specifically, when using cathode off-gas, containing both CH4 and H2, the produced CO2 by the MOB can be directly used by the HOB, and therefore result in an almost double COD conversion to protein compared to the use of raw biogas (i.e. 17% and 9% respectively). Furthermore, the synergetic relationship between MOB, autotrophic HOB and possibly heterotrophs, contained in the enriched cultures, decreases the risk of contamination from potentially pathogenic organisms, due to the limited available niches [46]. We investigated two approaches to use biogas to produce MP, namely the use of raw biogas, and the use of electrochemically upgraded gas streams. The direct use of biogas does not rely on the electrochemical production of H2 [38] or the use of natural gas [46]. On the other hand, the electrochemical upgrading results in the production of H2 in the cathode and O2 in the anode, therefore providing the additional advantage of producing H2 and O2. This H2 and O2 is then used by the HOB in combination with the use of the CO2 originating from the CH4 25

metabolism of the MOB, maximizing resource efficiency. In this approach, the gasses pass through the anolyte and catholyte solutions, that could deactivate potentially present microorganisms from the anaerobic digestion due to the extreme pH (<1.5 for anolyte and >10 for catholyte) [38]. This offers the advantage of providing a clean gas stream, compared to the biological biogas upgrading [39]. Although we delivered a proof of concept to produce microbial protein using electrochemically upgraded or raw real biogas, the process conditions should be improved to increase yields and kinetics. For instance, a continuous mode of cultivation would ensure higher product yields and production rates. This was illustrated by the study of Matassa et al. [22], who achieved yields of 0.07 and 0.28 g CDW g-1COD during sequential batch and continuous mode, respectively, corresponding with biomass production rates of 0.08 and 0.38 g CDW L-1 h-1.

5. Conclusions In the present study, CO2 and CH4 were successfully separated by electrochemical means, starting from real biogas stream, and then used for microbial protein production. Microbial protein was produced from the off-gas streams from electrochemical biogas upgrading units. The CH4 blended with H2 obtained electrochemically (cathode off-gas) showed the best results of microbial protein production in terms of protein content and microbial yield. This proof of concept demonstrated that electrochemical separation of biogas is capable to generate customized gas mixtures for enhanced microbial protein production.

26

6. Acknowledgments Nayaret Acosta was supported by the grant SENESCYT Convocatoria Abierta 2014 Primera Fase. Jo De Vrieze was supported as postdoctoral research fellow by the Research Foundation Flanders (FWO-Vlaanderen). Myrsini Sakarika was supported by the Catalisti cluster SBO project CO2PERATE, with the financial support of VLAIO (Flemish Agency for Innovation and Entrepreneurship), Cindy Ka Y Law was supported by from the ELECTRA project financed by the H2020 of the European Commission under Grant number GA 826244. The authors gratefully acknowledge Funda Torun for providing the pond sediment samples used as an inoculum for the HOB enrichment. We thank Emma Hernandez-Sanabria for useful suggestions and to Inka Vanwonterghem for critically reading the manuscript.

References [1] S. Kc, W. Lutz, The human core of the shared socioeconomic pathways: Population scenarios by age, sex and level of education for all countries to 2100, Global Environmental Change 42 (2017) 181-192. [2] D. Tilman, C. Balzer, J. Hill, B.L. Befort, Global food demand and the sustainable intensification of agriculture, Proc Natl Acad Sci U S A 108(50) (2011) 20260-20264. [3] A.J. Tanentzap, A. Lamb, S. Walker, A. Farmer, Resolving Conflicts between Agriculture and the Natural Environment, PLoS Biol 13(9) (2015) e1002242-e1002242. [4] L. Bouwman, K.K. Goldewijk, K.W. Van Der Hoek, A.H.W. Beusen, D.P. Van Vuuren, J. Willems, M.C. Rufino, E. Stehfest, Exploring global changes in nitrogen and phosphorus cycles in agriculture induced by livestock production over the 1900–2050 period, Proceedings of the National Academy of Sciences 110(52) (2013) 20882. [5] I. Pikaar, S. Matassa, K. Rabaey, B.L. Bodirsky, A. Popp, M. Herrero, W. Verstraete, Microbes and the Next Nitrogen Revolution, Environmental Science & Technology 51(13) (2017) 7297-7303. [6] I. Pikaar, S. Matassa, B.L. Bodirsky, I. Weindl, F. Humpenöder, K. Rabaey, N. Boon, M. Bruschi, Z. Yuan, H. van Zanten, M. Herrero, W. Verstraete, A. Popp, Decoupling Livestock from Land Use through Industrial Feed Production Pathways, Environmental Science & Technology 52(13) (2018) 7351-7359. [7] A. Ritala, S.T. Häkkinen, M. Toivari, M.G. Wiebe, Single Cell Protein—State-of-the-Art, Industrial Landscape and Patents 2001–2016, Frontiers in Microbiology 8(2009) (2017). [8] P. Miller, Billions of pounds of pumpkin will go to the landfill after Halloween. , 2018 (accessed 2019/04/19.2019). 27

[9] P. Grabowski, Reducing Waste and Harvesting Energy This Halloween. , 2013 (accessed 2019/04/19.2019). [10] X. Wang, G. Yang, Y. Feng, G. Ren, X. Han, Optimizing feeding composition and carbon–nitrogen ratios for improved methane yield during anaerobic co-digestion of dairy, chicken manure and wheat straw, Bioresource Technology 120 (2012) 78-83. [11] I.A. Nges, L. Björnsson, High methane yields and stable operation during anaerobic digestion of nutrient-supplemented energy crop mixtures, Biomass and Bioenergy 47 (2012) 62-70. [12] M. Wang, X. Zhang, J. Zhou, Y. Yuan, Y. Dai, D. Li, Z. Li, X. Liu, Z. Yan, The dynamic changes and interactional networks of prokaryotic community between co-digestion and mono-digestions of corn stalk and pig manure, Bioresource Technology 225 (2017) 2333. [13] P. Dwivedi, S.K. Dwivedi, M.C. Kalita, Biodiversity and Environmental Biotechnology, Scientific Publishers2007. [14] J. De Vrieze, J.B.A. Arends, K. Verbeeck, S. Gildemyn, K. Rabaey, Interfacing anaerobic digestion with (bio)electrochemical systems: Potentials and challenges, Water Research 146 (2018) 244-255. [15] G. Luo, S. Johansson, K. Boe, L. Xie, Q. Zhou, I. Angelidaki, Simultaneous hydrogen utilization and in situ biogas upgrading in an anaerobic reactor, Biotechnology and Bioengineering 109(4) (2012) 1088-1094. [16] K. Verbeeck, J. De Vrieze, M. Biesemans, K. Rabaey, Membrane electrolysis-assisted CO2 and H2S extraction as innovative pretreatment method for biological biogas upgrading, Chemical Engineering Journal 361 (2019) 1479-1486. [17] M.R. Martin, J.J. Fornero, R. Stark, L. Mets, L.T. Angenent, A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2, Archaea 2013 (2013) 11. [18] R. Muñoz, L. Meier, I. Díaz, D. Jeison, A review on the state-of-the-art of physical/chemical and biological technologies for biogas upgrading, 2015. [19] R. Kumar, L. Singh, A.W. Zularisam, F.I. Hai, Microbial fuel cell is emerging as a versatile technology: a review on its possible applications, challenges and strategies to improve the performances, International Journal of Energy Research 42(2) (2018) 369-394. [20] Z. Huang, L. Lu, D. Jiang, D. Xing, Z.J. Ren, Electrochemical hythane production for renewable energy storage and biogas upgrading, Applied Energy 187 (2017) 595-600. [21] R. Kumar, L. Singh, A.W. Zularisam, Exoelectrogens: Recent advances in molecular drivers involved in extracellular electron transfer and strategies used to improve it for microbial fuel cell applications, Renewable and Sustainable Energy Reviews 56 (2016) 13221336. [22] S. Matassa, W. Verstraete, I. Pikaar, N. Boon, Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria, Water Research 101 (2016) 137-146. [23] R. Whittenbury, K.C. Phillips, J.F. Wilkinson, Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria, Microbiology 61(2) (1970) 205-218. [24] K.A. Malik, D. Claus, Xanthobacter flavus, a New Species of Nitrogen-Fixing Hydrogen Bacteria, International Journal of Systematic and Evolutionary Microbiology 29(4) (1979) 283-287. [25] W.E. Balch, S. Schoberth, R.S. Tanner, R.S. Wolfe, Acetobacterium, a New Genus of Hydrogen-Oxidizing, Carbon Dioxide-Reducing, Anaerobic Bacteria, International Journal of Systematic and Evolutionary Microbiology 27(4) (1977) 355-361. 28

[26] B. Bowien, G.H. Schlegel, Physiology and Biochemistry of Aerobic HydrogenOxidizing Bacteria, 1981. [27] L. Joergensen, The methane mono-oxygenase reaction system studied by membraneinlet mass spectrometry, Biochemical Journal 225(2) (1985) 441. [28] P.J. Reeds, Dispensable and Indispensable Amino Acids for Humans, The Journal of Nutrition 130(7) (2000) 1835S-1840S. [29] A.E. Greenberg, L.S. Clesceri, A.D. Eaton, Standard Methods for the Examination of Water and Wastewater, 18 ed. ed., American Public Health AssociationAmerican Water Works Association, Water Environment Federation, Washington (D.C.), 1992. [30] S.J. Andersen, T. Hennebel, S. Gildemyn, M. Coma, J. Desloover, J. Berton, J. Tsukamoto, C. Stevens, K. Rabaey, Electrolytic Membrane Extraction Enables Production of Fine Chemicals from Biorefinery Sidestreams, Environmental Science & Technology 48(12) (2014) 7135-7142. [31] M.A.K. Markwell, S.M. Haas, L.L. Bieber, N.E. Tolbert, A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples, Analytical Biochemistry 87(1) (1978) 206-210. [32] A. Kokkoli, Y. Zhang, I. Angelidaki, Microbial electrochemical separation of CO2 for biogas upgrading, Bioresource Technology 247 (2018) 380-386. [33] X. Jin, Y. Zhang, X. Li, N. Zhao, I. Angelidaki, Microbial Electrolytic Capture, Separation and Regeneration of CO2 for Biogas Upgrading, Environmental Science & Technology 51(16) (2017) 9371-9378. [34] S.C. Baicu, M.J. Taylor, Acid–base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency, Cryobiology 45(1) (2002) 33-48. [35] Z. Rasouli, B. Valverde-Pérez, M. D’Este, D. De Francisci, I. Angelidaki, Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs, Biochemical Engineering Journal 134 (2018) 129-135. [36] B.T. Sheehan, M.J. Johnson, Production of bacterial cells from methane, Applied microbiology 21(3) (1971) 511-515. [37] L.M. Steinberg, R.E. Kronyak, C.H. House, Coupling of anaerobic waste treatment to produce protein- and lipid-rich bacterial biomass, Life Sciences in Space Research 15 (2017) 32-42. [38] M.E.R. Christiaens, S. Gildemyn, S. Matassa, T. Ysebaert, J. De Vrieze, K. Rabaey, Electrochemical Ammonia Recovery from Source-Separated Urine for Microbial Protein Production, Environmental Science & Technology 51(22) (2017) 13143-13150. [39] B. Khoshnevisan, P. Tsapekos, Y. Zhang, B. Valverde-Pérez, I. Angelidaki, Urban biowaste valorization by coupling anaerobic digestion and single cell protein production, Bioresource Technology 290 (2019) 121743. [40] L. Bongers, Energy generation and utilization in hydrogen bacteria, J Bacteriol 104(1) (1970) 145-151. [41] M. Øverland, A.-H. Tauson, K. Shearer, A. Skrede, Evaluation of methane-utilising bacteria products as feed ingredients for monogastric animals, Archives of Animal Nutrition 64(3) (2010) 171-189. [42] CME, Spotlight: How Trade Wars May Affect Ags Markets . 2019 2019/04/19). [43] P.J. Strong, M. Kalyuzhnaya, J. Silverman, W.P. Clarke, A methanotroph-based biorefinery: Potential scenarios for generating multiple products from a single fermentation, Bioresource Technology 215 (2016) 314-323. [44] T.S. Aas, B. Grisdale-Helland, B.F. Terjesen, S.J. Helland, Improved growth and 29

nutrient utilisation in Atlantic salmon (Salmo salar) fed diets containing a bacterial protein meal, Aquaculture 259(1) (2006) 365-376. [45] H.F. Schøyen, B. Svihus, T. Storebakken, A. Skrede, Bacterial protein meal produced on natural gas replacing soybean meal or fish meal in broiler chicken diets, Archives of Animal Nutrition 61(4) (2007) 276-291. [46] H. Bothe, K. Møller Jensen, A. Mergel, J. Larsen, C. Jørgensen, H. Bothe, L. Jørgensen, Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process, Applied Microbiology and Biotechnology 59(1) (2002) 33-39. [47] A. Ho, K. de Roy, O. Thas, J. De Neve, S. Hoefman, P. Vandamme, K. Heylen, N. Boon, The more, the merrier: heterotroph richness stimulates methanotrophic activity, The Isme Journal 8 (2014) 1945. [48] D. van der Ha, L. Nachtergaele, F.-M. Kerckhof, D. Rameiyanti, P. Bossier, W. Verstraete, N. Boon, Conversion of Biogas to Bioproducts by Algae and Methane Oxidizing Bacteria, Environmental Science & Technology 46(24) (2012) 13425-13431. [49] A. Ho, K. de Roy, O. Thas, J. De Neve, S. Hoefman, P. Vandamme, K. Heylen, N. Boon, The more, the merrier: heterotroph richness stimulates methanotrophic activity, The ISME Journal 8(9) (2014) 1945-1948. [50] T. Cumberlege, B. T., C. J., Assessment of environmental impact of FeedKind (TM) protein, UK, 2016. [51] R.S. Hanson, Ecology and Diversity of Methylotrophic Organisms, in: D. Perlman (Ed.), Advances in Applied Microbiology, Academic Press1980, pp. 3-39. [52] A.T. Bull, J.H. Slater, Microbial Interactions and Communities, Academic Press1982. [53] R. Schmaljohann, Oxidation of various potential energy sources by the methanotrophic endosymbionts of Siboglinum poseidoni (Pogonophora), Marine Ecology Progress Series 76(2) (1991) 143-148.

30

31

HIGHLIGHTS

Title: Microbial protein production from methane via electrochemical biogas upgrading. Nayaret Acosta1, Myrsini Sakarika1, Frederiek-Maarten Kerckhof1, Cindy Ka Y. Law1, Jo De Vrieze1 and Korneel Rabaey1 1Centre

for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links

653, B-9000 Gent, Belgium



Electrochemical upgraded biogas was used for microbial protein production.



CO2 and CH4 were used simultaneously as carbon sources in mixed cultures.



Upgraded cathode off-gas generates more microbial protein than raw biogas.

32